Eukaryotic organisms respond to environmental stimuli through an evolutionarily conserved signaling mechanism called the mitogen-activated protein kinase (MAPK) cascade. A typical MAPK pathway is composed of three protein kinases; MAPK, MAPK kinase (MAPKK) and MAPKK kinase (MAPKKK). Upon activation of the cascade in the cytoplasm, MAPK enters the nucleus to phosphorylate transcription factors, while MAPKK and MAPKKK appear to remain in the cytoplasm. This nuclear translocation of MAPK is important for proper gene expression in response to extracellular stimuli; however, it is not well understood how cells regulate the localization of MAPK. Because of the conservation of MAPK cascades through evolution, a genetically amenable model system, the fission yeast Schizosaccharomyces pombe, will be used to identify the determinants of the subcellular localization of each kinase, aiming to understand the molecular mechanisms of such specific localization in MAPK signaling. In S. pombe, the stress-responsive MAPKK, Wis1, is localized in the cytoplasm, while the downstream MAPK, Spc1 enters the nucleus in response to stress stimuli. For a more convenient way to observe protein localization, a S. pombe integration vector will be constructed that expresses an inserted gene as a GFP (green fluorescent protein) fusion. After treating cells with a stress stimulus, any change in localization will be visualized by fluorescence microscopy. These studies will shed light on the key step in MAPK signaling from the cytoplasm to the nucleus. It is expected that knowledge obtained in yeast is very informative to understand the regulation of human MAPK cascades, which play crucial roles in cellular proliferation and physiology. [unreadable] [unreadable]